1. Field of the Invention
The present invention relates to a method and apparatus for analyzing spectra obtained by a mass spectrometer and, more particularly, to a method and apparatus for analyzing spectra obtained by mass-analyzing ions to which molecules of a mobile phase solvent or impurities contained in the solvent are attached.
2. Description of Related Art
Where the masses of molecules (hereinafter referred to as sample molecules indicated by M) contained in a sample are analyzed using a mass spectrometer, the sample is ionized. Various methods are available for the ionization. In one of these methods, ions of the sample molecules with mobile phase solvent or impurities contained in the solvent attached to the sample molecules are generated by ionization.
A typical example of such ionization method is atmospheric pressure ionization (API) used as an interface between a liquid chromatograph (LC) and a mass spectrometer (MS).
Two kinds of API methods are available: electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). In either method, ionization is performed by movement of protons between sample molecules and molecules of a mobile phase solvent, such as methanol, acetonitrile, or acetic acid.
Where positive ions are detected by a mass spectrometer, protonated ions [M+H]+consisting of sample molecules M to which protons H+are attached are detected. Where negative ions are detected by a mass spectrometer, deprotonated ions [Mxe2x88x92H]xe2x88x92consisting of sample molecules M from which protons H+have been abstracted are detected.
If ions produced by API are only either protonated ions [M+H]+or deprotonated ions [Mxe2x88x92H]xe2x88x92, no serious problems take place. It is known, however, that adduct ions are generated in addition to protonated or deprotonated ions where molecules of mobile phase solvent or impurities contained in it are attached to sample molecules and that such adduct ions are detected by mass spectrometry and often appear in spectra.
Ions where molecules of a mobile phase solvent or impurities contained in it are attached to sample molecules as mentioned above are hereinafter referred to as impurity adduct ions. Impurity adduct ions, protonated ions, and deprotonated ions are collectively referred to as adduct ions. For example, in protonated ions, protons are adducts. In impurity adduct ions [M+NH4]+, ammonium ions are adducts as described later. Also, with respect to deprotonated ions, protons are conveniently referred to as adducts.
For example, where ionization is done by ESI using methanol as a mobile phase solvent and positive ions are detected by a mass spectrometer, it is empirically known that positive impurity adduct ions [M+NH4]+and/or [M+Na]+are sometimes detected in addition to protonated ions [M+H]+. In the ions [M+NH4]+, ammonium ions are attached to sample molecules M. In the ions [M+Na]+, sodium ions are attached to sample molecules M.
It is also empirically known that where methanol is used as a mobile phase solvent, ionization is performed by APCI, and positive ions are detected by mass spectrometry, positive impurity adduct ions [M+H+CH3OH]+where protons H+and methanol molecules are attached to sample molecules M are sometimes detected in addition to protonated ions [M+H]+.
Furthermore, it is experimentally known that where a sample is ionized by ESI using formic acid as a mobile phase solvent and negative ions are detected by mass spectrometry, negative impurity adduct ions [M+HCOO]xe2x88x92where formic acid ions are attached to sample molecules M are sometimes detected in addition to deprotonated ions [Mxe2x88x92H]xe2x88x92.
Where samples are ionized by API and mass analyzed in this way, peaks of impurity adduct ions appear in the resulting spectrum, in addition to peaks of protonated or deprotonated ions. This often makes it difficult to judge the molecular weight of the sample molecules based on the spectrum or to analyze the spectrum.
Accordingly, it is quite difficult to analyze spectra obtained by ionizing samples by API and mass analyzing them. Therefore, a rich amount of experience is necessary to determine the molecular weight of sample molecules based on the spectrum or to analyze the spectrum. In addition, there is even the problem that the results of spectral analysis differ according to the degree of experience of each analyst.
The case where ionization is performed by API has been described thus far. Impurity adduct ions may also be generated where ionization is performed by methods other than API, e.g., chemical ionization (CI), fast atom bombardment (FAB), matrix assisted laser desorption (MALDI), and field desorption (FD). Where mass spectra obtained by these ionization methods are analyzed, similar problems take place. This is a first problem that the present application tackles.
Spectra obtained by ionizing samples by the aforementioned various ionization methods and mass analyzing them are compared with a very large number of mass spectra registered in a commercially available library, or database, of mass spectral data to identify the chemical formulas of observed ions. However, such registered mass spectra have all been derived by electron impact (EI) ionization that is a hard ionization method. Its feature is that the peaks of each individual molecular ion [M]+charged positively by release of one electron from each sample molecule are distributed in the highest mass-charge ratio (m/z) region of the resulting spectrum while peaks of fragment ions produced by fragmentation of molecular ions [M]+are distributed in a lower m/z region than the molecular ions. One example is a mass spectrum of toluene as shown in FIG. 1. This spectrum is a bar-type spectrum in which the peaks of a measured mass spectrum are data processed and represented as a bar graph. That is, each peak is represented in terms of a bar.
Such a library, or database, of mass spectra owing to EI is generally applied to mass spectra obtained by a GC/MS instrument that is a combination of a gas chromatograph and a mass spectrometer. In a GC/MS measuring system, peaks of adduct ions are not contained at all in mass spectra. Consequently, where samples ionized by soft ionization methods, such as API, CI, FAB, MALDI, and FD are compared with mass spectra which are obtained by a mass spectrometer and contain many peaks of adduct ions, pattern mismatch occurs frequently, even if they are mass spectra of the same compound.
Where samples are ionized by El, molecular ions [M]+that are charged positively by release of one electron from each sample molecule are observed routinely. However, mass spectra obtained by ionizing samples by a soft ionization method such as API, CI, FAB, MALDI, or FD and detecting the resulting ions by a mass spectrometer contain almost no such molecular ions [M]+.
Where API, CI, FAB, MALDI, or FD is used, fragment ions are not readily produced because of a soft ionization method. Yet, fragment ions can be produced by ESI or APCI by using in-source CID or in-source fragmentation that produces fragment ions by applying a voltage of tens of volts to the ion introduction port (orifice) of the vacuum region from the atmospheric-pressure ionization region to momentarily accelerate ions for collision with atmospheric gas.
FAB is a somewhat harder ionization method than ESI and APCI and, therefore, fragment ions are often produced depending on the nature of the measured compound.
Where mass spectra are obtained by a soft ionization method as described, if the measurement is performed by in-source CID, spectra having many fragment ions can be derived. Yet, there is the problem that the hit (match) rate is low when a search is done, because the patterns are widely different from those of mass spectra of the same compounds registered in commercially available libraries, or databases.
The reason why the hit rate is so low lies in the existence of adduct ions, such as [M+H]+, [M+Na]+, [M+NH4]+, and [M+H+solvent]+which are observed in LC/MS mass spectra obtained by a soft ionization method and which are in a higher mass region than [M]+ions. One example is a mass spectrum of reserpine as shown in FIG. 2. Therefore, it is required to construct a new general-purpose database that is dedicated for LC/MS applications and different from databases for GC/MS applications. However, mass spectra obtained by LC/MS using ion source structures fabricated by different instrument manufactures are different somewhat from each other because the source structures are subtly different from each other.
Accordingly, it can be hardly expected that an LC/MS database is constructed which has a high degree of generality and can be applied to instruments of all manufactures such as the existing GC/MS libraries. Nonetheless, it is not realistic to construct a database made up of tens of thousands of items for each manufacturer if time and labor are considered. This is a second problem that the present application tackles.
Accordingly, in view of the foregoing, it is a first object of the present invention to provide a novel method and apparatus permitting one to easily analyze mass spectra containing peaks of impurity adduct ions without skill.
It is a second object of the present invention to provide a novel method and apparatus for analyzing LC/MS mass spectra obtained by a soft ionization method, based on a commercially available, general-purpose GC/MS database constructed by EI.
A first method of analyzing a mass spectrum in accordance with the present invention achieves the above-described first object and comprises the steps of: comparing entered information about an ionization method, a detection polarity used by a mass spectrometer, and a mobile phase solvent with information registered in a database portion about ionization methods, detection polarities used by the mass spectrometer, mobile phase solvents, and adduct ions expected to be detected under these conditions; detecting peaks of adduct ions contained in data detected by the mass spectrometer; and determining the molecular weight of sample molecules based on the detected mass-to-charge ratios of the adduct ions.
In one feature of this method, the database portion holds information about ionization methods, detection polarities used by the mass spectrometer, mobile phase solvents, adduct ions expected to be detected under these conditions, and their names or appellations in such a way that at least one of these items can be added or rewritten.
In another feature of this method, the detection of the peaks of the adduct ions is performed based on whether the difference in mass-to-charge ratio (m/z) between at least two peaks observed in a high m/z region is coincident with the difference in mass-to-charge ratio between at least two species of adduct ions having the possibility of being observed by a measuring system.
In a further feature of this method, the mass spectrum is created by a soft ionization method.
In yet another feature of this method, the soft ionization method is one selected from the group consisting of API, CI, FAB, MALDI, and FD.
In still another feature of this method, the API is one of ESI and APCI used as an interface between a liquid chromatograph and the mass spectrometer.
First apparatus for analyzing a mass spectrum in accordance with the present invention comprises: an input portion for entering information about an ionization method, a detection polarity used by a mass spectrometer, and a mobile phase solvent; a database portion having registered information about ionization methods, detection polarities used by the mass spectrometer, mobile phase solvents, and adduct ions having the possibility of being detected under these conditions, for comparison with the information entered from the input portion; and a control portion for detecting peaks of adduct ions within data detected by the mass spectrometer based on the results of the comparison between the information entered from the input portion and the information registered in the database portion and determining the molecular weight of sample molecules based on the detected mass-to-charge ratios of the adduct ions.
In one feature of this apparatus, the database portion holds information about ionization methods, detection polarities used by the mass spectrometer, mobile phase solvents, adduct ions expected to be detected under these conditions, and their names or appellations in such a way that at least one of these items can be added or rewritten.
In another feature of this apparatus, detection of the peaks of the adduct ions is performed based on whether the difference in mass-to-charge ratio (m/z) between at least two peaks observed in a high m/z region is coincident with the difference in mass-to-charge ratio between at least two species of adduct ions having the possibility of being observed by the measuring system.
In a further feature of this apparatus, the mass spectrum is created by a soft ionization method.
In yet another feature of this apparatus, the soft ionization method is one selected from the group consisting of API, CI, FAB, MALDI, and FD.
In still another feature of this apparatus, the API is one of ESI and APCI used as an interface between a liquid chromatograph and the mass spectrometer.
A second method of analyzing a mass spectrum in accordance with the present invention comprises the steps of: comparing entered information about an ionization method, a detection polarity used by a mass spectrometer, and a mobile phase solvent with information registered in a database about ionization methods, detection polarities used by the mass spectrometer, mobile phase solvents, and adduct ions expected to be detected under these conditions; detecting peaks of adduct ions contained in data detected by the mass spectrometer; deleting the peaks of the adduct ions from the detected data; adding peaks of a given height to peak positions of molecular ions corresponding to the adduct ions; and then searching the detected data against the database.
In one feature of this method, the database holds information about ionization methods, detection polarities used by the mass spectrometer, mobile phase solvents, adduct ions expected to be detected under these conditions, and their names or appellations in such a way that at least one of these items can be added or rewritten.
In another feature of this method, the detection of the peaks of the adduct ions is performed based on whether the difference in mass-to-charge ratio (m/z) between at least two peaks observed in a high m/z region is coincident with the difference in mass-to-charge ratio between at least two species of adduct ions having the possibility of being observed by the measuring system.
In a further feature of this method, when an operation for deleting the peaks of the adduct ions from the detected data and adding the peaks of the given height to the peak positions of the molecular ions corresponding to the adduct ions is performed, deletion and addition of isotope peaks are performed simultaneously.
In a still further feature of this method, the given height corresponds to the total sum of the peaks of the deleted adduct ions.
In an additional feature of this method, the mass spectrum is created by a soft ionization method.
In yet another feature of this method, the soft ionization method is one selected from the group consisting of API, CT, FAB, MALDI, and FD.
In still another feature of this method, the API is one of EST and APCI used as an interface between a liquid chromatograph and the mass spectrometer.
A second apparatus for analyzing a mass spectrum in accordance with the present invention comprises: an input portion for entering information about an ionization method, a detection polarity used by a mass spectrometer, and a mobile phase solvent; a database portion holding registered information about ionization methods, detection polarities used by the mass spectrometer, mobile phase solvents, and adduct ions having the possibility of being detected under these conditions, for comparison with the information entered from the input portion; and a control portion for detecting peaks of adduct ions within data detected by the mass spectrometer based on the results of the comparison between the information entered from the input portion and the information registered in the database portion, deleting the peaks of the adduct ions from the detected ions, adding peaks of a given height to peak positions of molecular ions corresponding to the adduct ions, and searching the detected data against the database.
In one feature of this method, the database portion holds information about ionization methods, detection polarities used by the mass spectrometer, mobile phase solvents, adduct ions expected to be detected under these conditions, and their names or appellations in such a way that at least one of these items can be added or rewritten.
In another feature of this apparatus, the detection of the peaks of the adduct ions is performed based on whether the difference in mass-to-charge ratio (m/z) between at least two peaks observed in a high m/z region is coincident with the difference in mass-to-charge ratio between at least two species of adduct ions having the possibility of being observed by the measuring system.
In a further feature of this apparatus, when an operation for deleting the peaks of the adduct ions from the detected data and adding the peaks of the given height to the peak positions of molecular ions corresponding to the adduct ions is performed, deletion and addition of isotope peaks are performed simultaneously.
In yet another feature of this apparatus, the given height corresponds to the total sum of the deleted adduct ions.
In a still further feature of this method, the mass spectrum is created by a soft ionization method.
In yet another feature of this method, the soft ionization method is one selected from the group consisting of API, CI, FAB, MALDI, and FD.
In still another feature of this method, the API is one of ESI and APCI used as an interface between a liquid chromatograph and the mass spectrometer.
Other objects and features of the invention will appear in the course of the description thereof, which follows.